Introduction: Lowvitamin D status (defined by serum 25(OH)D levels and low sunlight exposure) are known environmental risk factors for the development of multiple sclerosis (MS). Add-on Vitamin D supplementation trials in established MS have been inconclusive. The effects of vitamin D supplementation to prevent MS development in high-risk clinically isolated syndrome (CIS) are unknown. Objectives and Aims: To determine whether vitamin D supplementation in patients with CIS and positive MRI (modified from Paty’s criteria)delays time to either new clinical or radiological activity. Methods: In this double-blind trial,eligible participants were randomised 1:1:1:1 to placebo, 1000, 5000, or 10000 IU of oral vitamin D3 daily within each study site (n=23) and followed for up to 48 weeks across academic MS centres in Australia & New Zealand. Between 2013 and 2020, we enrolled 204 participants. Brain MRI scans were performed at baseline, 24 and 48 weeks.The primary outcomewas time to MS conversion (2010 McDonald criteria) i.e. confirmed relapse or new Brain MRI T2 lesion. Two co-primary analyses were undertaken:1. Outcome based on allocated dosage of vitamin D or placebo and 2. Outcome based on measured serum 25(OH)D levels during the study. Results: n the intention-to-treat analysis based on assigned dose, the Odds Ratios (ORs), with 95% Confidence Intervals (CI) for conversion to definite MS were placebo 1.00 (reference), 1000 IU 0.94 (0.41, 2.19), 5000 IU 1.58 (0.67, 3.71) and 10000 IU 1.52 (0.64, 3.59). The as-measured serum 25(OH)D analysis (co-primary outcome) was not associated with conversion.A fully adjusted model including latitude, site, smoking, age, sex and baseline measures ofmono or polysymptomatic onset, presence of infra-tentorial lesions, and prior use of steroids produced ORs (95% COIS) for conversion in the assigned dose analysis of placebo 1.00 (reference), 1000 IU 0.48 (0.18, 1.27), 5000 IU 2.49 (1.02, 6.08), and 10000 IU 1.10 (0.40, 3.04). Conclusions: This double-blind placebo-controlled randomised study of vitamin D supplementation in high risk CIS indicates that vitamin D supplementation is not an effective treatment to prevent development of relapsing-remitting MS.